Measurement of Hepcidin-25 in Serum Using the Agilent 6490 Triple Quadrupole LC/MS with ifunnel Technology

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1 Measurement of Hepcidin-25 in Serum Using the Agilent 6490 Triple Quadrupole LC/MS with ifunnel Technology Application Note Clinical Research Authors Christophe Hirtz, Constance Delaby, Pauline Bros, Jérôme Vialaret, and Sylvain Lehmann CHU Montpellier, Institut de Recherche en Biothérapie, Hôpital St Eloi, Laboratoire de Biochimie Protéomique Clinique and CCBHM, Montpellier, France Abstract A novel nano HPLC/MS method has been developed for the rapid, robust, reliable, and accurate determination of hepcidin-25 levels in human serum. It relies on straightforward pre-analytical steps that are cost effective, avoiding the need for solid phase extraction. This Application Note demonstrates how nano HPLC/MS can be used to deliver better reproducibility and a limit of detection (LOD) for hepcidin less than half that of current C-ELISA assays.

2 Introduction Both intestinal iron absorption and macrophage iron recycling are regulated by hepcidin, a 25 amino-acid (aa) cysteine-rich peptide first characterized in Isoforms of the peptide (20, 22, and 24-amino acids) also exist 4, but their clinical relevance is unknown. A number of methods have been developed for this purpose using multiple technologies, including radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and more recently, mass spectrometry analysis (SELDI-TOF and LC/MS/MS) 7 9. The immunoassays employ antihepcidin antibody, with the potential to recognize the different isoforms. The methods based on mass spectrometry can specifically detect all the hepcidin isoforms, and they can provide absolute quantitation through the use of a spiked internal standard. All of these methods have demonstrated limited specificity and reproducibility as well as lack of concurrence in quantified levels of hepcidin, across the assay technologies. In an effort to resolve these issues, the different methods have been recently compared through round robin studies 7,8. The results demonstrated that development of a reliable assay remains quite challenging. The use of an internal standard was recommended for mass spectrometry based methods. These studies also called for development of a calibrator mimicking human serum and development of a consensus on calibrator levels. This Application Note describes a recently published liquid chromatography tandem-mass spectrometry (LC/MS/MS) analytical method using a nano HPLC-chip on the Agilent 6490 Triple Quadrupole LC/MS for the reliable quantification of hepcidin-25 in human serum 10. The method was evaluated for specificity, reproducibility, repeatability, and determination of LODs, limits of quantitation (LOQs), and linearity. These results were compared to the well-characterized C-ELISA method used to quantify serum hepcidin-25 in human samples 11. Although discrepancies were observed in the absolute values of hepcidin measured, there was good correlation between the two approaches (R 2 = 0.96). 2

3 Experimental Reagents and standards Reagents were obtained as described 10. The human endogenous hepcidin standard (DTHFPICIFCCGCCHRSKCGMCCKT) and the internal hepcidin standard (DTHFPICIFCCGCCHRSKCGMCCKT) [ 13 C 6, 15 N 4 ] Arg 16 were purchased from Eurogentec (Seraing, Belgium) with a purity > 97 % assessed by reverse phase HPLC and mass spectrometry. Samples The serum samples included in this study were part of a biobank (officially registered # DC ) and all participants gave their informed consent to use their sample for research conducted in accordance with the local ethics committee. Instruments This method was developed using an Agilent 1290 Infinity HPLC-Chip/MS System coupled to an Agilent 6490 Triple Quadrupole LC/MS equipped with a nano ESI source. The instrument conditions are listed in Table 1. Sample preparation Serum samples (50 µl) were extracted using trichloroacetic acid as described 10. Data analysis Agilent MassHunter Quantitative Analysis Software was used for data treatment. For the calibration curve, all replicates results were loaded into the software database, and the automatic quantification method was then used to obtain the appropriate calibration curve type with the best fit to the experimental data. Statistical analyses were performed using MedCalc (7.3) software. Bland and Altman 12 and Deming adjusted regression curves 13 were used to test the commutability of the methods. Table 1. HPLC-Chip and MS instrument conditions for the hepcidin quantification method. LC conditions HPLC-Chip ProtID-Chip-43 (II) 43 mm 300 Å C18 chip with 40 nl trap column (p/n G ) Injection volume 4 µl in 15 % B, followed by 7 µl of flush volume Mobile phase A = 97:3:1 v/v/v (water/acetonitrile/formic acid) B = 10:90:1 v/v/v (water/acetonitrile/formic acid) Run time 9 minutes Flow rate 2.5 µl/min for sample loading, 0.6 µl/min for separation Gradient program 7 minutes linear gradient from 3 % to 100 % B, then 2 minutes wash (100 % B) and 1 minute equilibration (97 % A) MS conditions Mode ESI mode, positive ionization; MRM Capillary tension 1,700 2,100 V Nebulizer gas flow 11 L/min Nebulizer gas temperature 150 C Cell accelerator voltage 4 V Delta EMV 200 V Fragmentor voltage 380 V Dwell time 40 msec MS1 Wide MS2 Wide Ion funnel voltage pressure Low: 80 V; High: 180 V Acquisition parameters The acquisition parameters used in the multiple reaction monitoring (MRM) method for both endogenous hepcidin and the isotopically-labeled hepcidin internal standard are shown in Table 2. Table 2. Acquisition parameters for the hepcidin MRM method. Precursor ion (m/z) Product ion (m/z) Collision energy (ev) Endogenous hepcidin Isotopically-iabeled hepcidin internal standard 1, ,2 11 Quantifier ion; all others are qualifier ions. 3

4 Results and Discussion Analytical validation A typical total ion chromatogram and extracted ion chromatograms using MRM are shown in Figure 1 for serum spiked with endogenous hepcidin standard and isotopically-labeled hepcidin standard. Calibration curves were constructed in MassHunter software over the range ng/ml, based on hepcidin quantification values determined using orthogonal analysis methods. The calibration curve equation was linear (y = x ) when the origin was ignored and the equation was weighted 1/y. The calibration coefficient (R 2 ) was (Figure 2). The specificity of the method was confirmed by the absence of a hepcidin-25 peak at the appropriate retention time in blank samples (normal goat serum containing 0 ng/ml of hepcidin-25). Analysis of four replicates of one calibration curve point on the same day was used to evaluate intra-assay precision and accuracy. The intra-assay precision was 7 % at 50 ng/ml and 28 % at 5 ng/ml. The intra-assay accuracy was approximately 100 % at 20, 50, 100, and 200 ng/ml and almost 80 % at 5 and 10 ng/ml (Table 3). Inter-assay precision and accuracy were evaluated by quantifying the 50 and 200 ng/ml calibration standards on four separate days. Inter-assay precision was around 9 10 %, and the accuracy was close to 80% at both concentrations. The LOD and LOQ were 2 ng/ml and 6 ng/ml, respectively. Total ion chromatogram (TIC) and extracted ion chromatogram (XIC) of hepcidin-25 in serum samples using nano HPLC-MS 10 4 Hepcidin peak Relative response TIC on serum sample MRM endogenous hepcidin (559 & 694) MRM labeled hepcidin (560 & 696) y = x R 2 = Figure 1. Typical TIC and extracted ion chromatograms (EICs) for endogenous hepcidin standard and isotopically-labeled hepcidin spiked into human serum Relative concentration Figure 2. Calibration curves of hepcidin-25 using model matrix (normal goat serum) in the concentration range of 0 to 500 ng/ml (0, 5, 10, 20, 50, 100, 200, ng/ml). The calibration equation was linear when ignoring origin and weighted 1/y. R 2 = Table 3. Intra-assay coefficient of variation and accuracy (%). Hepcidin-25 concentration (ng/ml) Intra-assay coeffcient of variation (% CV) Intra-assay accuracy (%) 4

5 Comparison to competitive ELISA Serum samples previously quantified using the C-ELISA method 11 were analyzed using this nano HPLC/MS MRM method. A positive significant correlation with R 2 = 0.89 and Pearson coefficient P < (Figure 3) However, the slope of the linear correlation (5,0255x) indicates that the MRM assay provided values five times lower than those obtained with the C-ELISA. This result is likely related to the fact that the MS methods quantify only the 25 aa isoform and, therefore, are fully specific. The difference may also be due in part to the differences in the origin and purity of the material used for the standard curves in each of the two methods. Hepcidin concentration by ELISA (ng/ml) y = x R 2 = Hepcidin concentration by MRM (ng/ml) Figure 3. Comparison of hepcidin-25 concentrations measured in serum samples using nano HPLC/MS (MRM) and ELISA methods. A positive significant correlation has been obtained with an R 2 value of 0.89 and a Pearson coefficient P < This nano HPLC/MS method generated a coefficient of variation (CV) of only 9 % for 30 participants, while the CV for the C-ELISA study described by Ganz et al. ranged from 5 19 % 11. The LOD for the nano HPLC/MS method is also less than half of the LOD determined for the C-ELISA method, at 2 ng/ml, versus 5.5 ng/ml. We have, therefore, developed a robust and reliable analytical method for hepcidin-25 suitable for quantification of hepcidin-25 in human serum. Conclusions This nano HPLC/MS method developed on the Agilent 6490 Triple Quadrupole LC/MS is suitable for the accurate, robust, and reproducible measurement of hepcidin in human serum samples. It relies on straightforward pre-analytical steps that are cost effective and avoid the need for solid phase extraction. While the obtained hepcidin levels differed from those determined using the reference C-ELISA assay, this nano HPLC/MS method has the advantage of being able to detect and quantify the truncated 20, 22, and 24-amino acid isoforms of hepcidin, whose clinical relevance is not yet known. 5

6 References 1. C.H. Park, et al. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J. Biol. Chem. 276, (2001). 2. C. Pigeon, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload J. Biol. Chem. 276, (2001). 3. G. Nicolas, et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice Proc. Natl. Acad. Sci. USA 98, (2001). 4. B. Rochat, et al. Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform Bioanalysis 5, (2013). 5. J.J. Kroot, et al. Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders Clin. Chem. 56, (2010). 6. J.J. Kroot, et al. Hepcidin in human iron disorders: diagnostic implications Clin. Chem. 57, (2011). 7. J.J. Kroot, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization Haematologica 94, (2009). 8. J.J. Kroot, et al. Second round robin for plasma hepcidin methods: first steps toward harmonization Am. J. Hematol. 87, (2012). 9. C. Delaby, C. Beaumont, Serum hepcidin assay in 2011: where do we stand? Ann. Biol. Clin. (Paris) 70, (2012). 10. C. Delaby, et al. EuPA Open Proteomics, Article in revision. 11. T. Ganz, et al. Immunoassay for human serum hepcidin Blood 112, (2008). 12. J.M. Bland, D.G. Altman Statistical methods for assessing agreement between two methods of clinical measurement Lancet 1 (8476), (1986). 13. P.J. Cornbleet, N. Gochman Incorrect least-squares regression coefficients in method-comparison analysis Clin. Chem. 25, (1979). 14. B.J. Sasu, et al. Serum hepcidin but not prohepcidin may be an effective marker for anemia of inflammation (AI) Blood Cells Mol. Dis. 45, (2010). For Research Use Only. Not for use in diagnostic procedures. This information is subject to change without notice. Agilent Technologies, Inc., 2014 Published in the USA, August 5, EN